Metabolomic Profiling Identifies Mechanisms Regulating Hypoxia-Induced Drug Resistance In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 121-121
Author(s):  
Patricia Maiso ◽  
Daisy Huynh ◽  
Yosra Aljawai ◽  
John M. Asara ◽  
Antonio Sacco ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Drug Resistance ◽  
Energy Metabolism ◽  
High Risk ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Hypoxic Conditions

Abstract Background Multiple Myeloma (MM) is the second most prevalent hematological malignancy and remains incurable, with a median survival of 3-7 years. However, despite the success of the new treatments, most patients still succumb to their disease. In about 20-25% of high-risk patients, MM progresses rapidly and does not respond to conventional therapies leading to rapid extramedullary disease and demise of these patients. One such regulator of dissemination and drug resistance is the dynamic process of oxygen deprivation or hypoxia. A number of studies show that hypoxia promotes neo-angiogenesis, cancer progression, epithelial-mesenchymal transition (EMT), acquisition of metastasis potential and stem-cell features, as well as resistance to therapy by activating adaptive transcriptional programs. Targeting hypoxia, and the metabolic pathways regulated by hypoxia in the tumor cells, could lead to novel opportunities for cancer therapy. Rapidly proliferating hypoxic cancer cells undergo a “metabolic switch” to anaerobic glycolysis. This altered energy metabolism has been shown to be associated with activated oncogenes and mutant tumor suppressors, which are more prevalent in patients with high-risk MM. We aimed 1) to examine the role of HIF-1a and HIF-2ain regulating drug resistance in vitro and in vivo and 2) to identify specific hypoxia-regulated genes and regulators of energy metabolism leading drug resistance in MM. Methods The effect of hypoxia was analyzed in different MM cell lines (MM1S, RPMI8226, U266 and H929) in basal conditions and after the treatment with bortezomib, dexamethasone or melphalan. The cytotoxicity was analyzed by means of MTT assay. Apoptosis studies were performed by flow cytometry. Gene expression profile of MM1S cells treated with bortezomib was compared in normoxia vs hypoxia using D-chip and GSEA softwares. Genes with expression changes greater or lower than 2 fold in either direction were selected. HIF1A and HIF2A knockdowns were performed in MM1S using lentiviral vectors. For metabolite collection, samples were re-suspended using HPLC grade water for mass spectrometry and analyzed using a 5500 QTRAP hybrid triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC system (Shimadzu). A total of 254 endogenous water soluble metabolites were analyzed. Results We observed that MM cell lines were resistant to bortezomib and melphalan in hypoxic conditions (12 hours at 0.5% of oxygen levels) compared to normoxic conditions. At transcriptional and protein level. cells treated with bortezomib in hypoxic conditions affected a large number of genes/proteins involved in cell cycle such us p21, p53 and p57, cell death and glucose metabolism. However, cell cycle arrest was not responsible for the resistance of MM cells to bortezomib that was observed in hypoxic conditions. Therefore, we investigated mechanisms that are mediated by hypoxia and can regulate drug resistance. HIF1A knockdown restored the effect of bortezomib in MM1S and increased the percentage of apoptosis in cells treated with bortezomib under hypoxic conditions. To further explore the role of hypoxia in the regulation of tumor metabolism downstream of HIF1A, metabolomic studies were performed to characterize metabolic alterations following bortezomib treatment in hypoxic and normoxic conditions. This analysis revealed that hypoxic tumor cells treated with or without bortezomib show significant metabolic changes involving multiple pathways, the most significant of which are intermediates in glucose metabolism such us glucose-6-phosphate, fructose-6-phosphate, 3-phosphoglycerate and phosphoenolpyruvate. We also observed a decrease in measured tricarboxylic acid cycle (TCA) cycle intermediates (citrate, fumarate and malate) after hypoxia exposure and a significant increase of LDHA levels. We assessed the metabolic response to several drugs and shRNAs targeting different glycolytic enzymes (HK2, PFKBP3, PFKBP4 and LDHA). Of these, the most significant changes were observed with LDHA knockdown where these overcame resistance to bortezomib in hypoxic conditions. Conclusion Hypoxic conditions are essential for drug resistance and glucose utilization. These data provide new therapeutic targets and associated biomarkers for the treatment of Multiple Myeloma. Disclosures: Ghobrial: Onyx: Membership on an entity’s Board of Directors or advisory committees; BMS: Membership on an entity’s Board of Directors or advisory committees; BMS: Research Funding; Sanofi: Research Funding; Novartis: Membership on an entity’s Board of Directors or advisory committees.

scholarly journals Role of High-Dose Melphalan and Autologous Stem Cell Transplantation in Multiple Myeloma Patients Presenting with t(11;14)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4580-4580 ◽  
Author(s):  
Eduardo Sobejano ◽  
Veronica Gonzalez De La Calle ◽  
Victor Higuero ◽  
Fernando Escalante ◽  
Ramón García-Sanz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
High Risk ◽  
Board Of Directors ◽  
Research Funding ◽  
University Hospital ◽  
First Line ◽  
Advisory Committees ◽  
Baseline Characteristics ◽  
Standard Risk

INTRODUCTION The t (11; 14) by fluorescent in situ hybridization (FISH) is found in 15-20% of patients with multiple myeloma (MM) . Although it was classically considered a standard risk translocation or even a good prognosis, recent studies conducted in the era of new drugs show contradictory results and it is not well established if they have to be considered intermediate or standard risk. The possibility of using targeted therapy with venetoclax for patients harboring t(11;14) makes the investigation of the outcome of newly diagnosed multiple myeloma (NDMM) with t(11;14) as relevant. METHODS We analyzed the baseline characteristics and outcome of patients with t(11;14)and receiving HDT-ASCT within the series of 647 patients with NDMM between 1988 and 2018 according to the current criteria at each moment at two academic hospitals in Spain (University Hospital of Salamanca and Hospital of Leon) . The FISH was performed on selected cells according to international regulations and centralized at the University Hospital of Salamanca. For this purpose, a descriptive cross-sectional study was first conducted comparing the characteristics of patients with t (11; 14) versus the rest. The final objective wasto evaluate the role of HDT-ASCT in NDMM with t(11;14). RESULTS The baseline characteristics of the whole series were: a median age of 71years (yrs) (range:30-96). 217 patients (33,5%) were under 65 years. 352 (56.2%) were IgG; 161 (25.7%) IgA; 87 (13.9%) Bence Jones; 19 (3%) non-secretors, and 5 and 2 cases were IgD and IgM, respectively. 320 (53.2%) received novel agents as part of the first line of therapy. Overall, 153 (27.8%) achieved complete response (CR) after first line, and 403 (73.1%) at least a partial response. After a median follow-up for living patients of 4.26 yrs (range: 0,1-27.3), the OS of the entire series was 2.74 years. T(11;14) was performed in 440 NDMM patients and was positive in 80 (18.2%). Only in 5 patients other high-risk alterations (t (14:16), t (4:14) or del17p (p53)) were detected. The baseline characteristics of patients with and without t (11:14) did not show significant differences, except for the heavy chain pattern(p <0,001). IgA was lower in patients with t(11:14) 12,8% (10 out of 78)vs 27,7% (98 out of 353). Of note, most patients with non-secretory MM (10 out of 16, 62,5%) had the t(11;14) whilst in the conventional secretory MM patients, t(11;14) was observed in 68out of 415(16,4%). In addition, the plasma cell bone marrow infiltration was significantly higher in patients with t(11;14)(> 60% Plasma Cells) 32.8% vs 13.3%(p <0.001)). HDT-ASCT was performed in 162 patients (25%)and 22 of them (13,5%) were positive for the t(11:14) and only in 2 patients, other high-risk alterations were detected.The induction therapy received in both treatments arms was homogeneous basically consisted on combinations of proteasome inhibitors plus immunomodulatory drugs. The median OS for NDMM patients undergoing ASCT was 4,33 years. (range: 0,47-26,85) and the median PFS for this patients was 2,25 yrs (range: 0,1-27,25) The median PFS for patients with t (11/14) undergoing ASCT trended to be higher than that observed in patients without t(11;14) who received also HDT-ASCT (99.1 vs 54.9 months), without obtaining significant results, (p 0.205) maybe due to the small number of patients (Figure 1).The median OS in the group of patients with and without t(11:14) undergoing ASCT was 120,8 vs 140 months (p= 0,829). In the cohort of non eligible ASCT patients both median PFS and OS for patients with t(11:14) was similar than that observed in patients without t(11:14)(median PFS of 19,9 vs 19,4 months) (p 0,438) and (median OS of 31,5 vs 44 months) (p 0,424), respectively. CONCLUSION T(11;14) seems to be a cytogenetic abnormality more frequently observed in patients with NDMM and non secretory phenotype what requires further investigation. Patients with t(11;14) benefit the most if they received HDT-ASCT and it would represent a therapeutic strategy of choice if the patient is transplant-eligible. Figure 1 Disclosures Puig: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding; The Binding Site: Honoraria; Takeda, Amgen: Consultancy, Honoraria. Mateos:Abbvie: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Pharmamar: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria; EDO: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals The Lysine-Specific Demethylase KDM4A/JMJD2A Acts As a Tumor Suppressor in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 191-191
Author(s):  
Fengyan Jin ◽  
Shaji K. Kumar ◽  
Yun Dai
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Tumor Suppressor ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Committees ◽  
Apoptotic Genes

Abstract Introduction: Histone lysine methylation, a reversible event dynamically and reciprocally regulated by lysine methyltransferases (KMTs) and demethylases (KDMs), represents one of the major epigenetic mechanisms for regulation of chromatin remodeling and gene expression re-programming. The KDM4 family, which belongs to the Jumonji C (JmjC)-domain-containing proteins (JMJDs), consists of five members, including KDM4A-E that demethylate H3K9me2/3 and/or H3K36me2/3 in a Fe2+- and α-ketoglutarate-dependent manner. KDM4 proteins are involved in various cellular processes such as gene transcription and translation, DNA replication, DNA repair, apoptosis, and stem cell renewal. Notably, increasing evidence implicates KDM4 dysregulation in promoting genomic instabilities and oncogenesis, thereby which is considered as a potential target for emerging cancer epigenetic therapy. Although KDM4A, a member of the KDM4 family, has been widely studied in many solid tumors including breast, prostate, bladder cancer, its role in hematopoietic malignancies, including multiple myeloma (MM), remains unknown. Materials and Methods: Human MM cell lines (U266, RPMI8226, H929, OPM-2) were employed. After exposed to hypoxia (or the chemical hypoxia mimetic lactic acid) and anti-MM agents (e.g., bortezomib/Btz), cells were analyzed by flow cytometry, qPCR, Western blot to monitor apoptosis, cell cycle, proliferation (Ki67), DNA double-strand break/DSB (γH2A.X), expression of 1q21 and anti-apoptotic genes, as well as activation of the NF-κB and HIF pathways. The shRNA approach was used to knock down KDM4A for functional evaluation. The findings from in vitro experiments involving cell lines were then validated in primary MM samples to link KDM4A expression to disease progression and therapeutic response. Results: Analysis of the MM genome-wide GEP databases revealed that KDM4A mRNA was significantly up-regulated in MGUS and MM, but not SMM, compared to normal control, as well as in relapsed MM, compared to newly-diagnosed MM. To our surprise, KDM4A expression rather favored overall survival of MM patients, including those carrying 1q21 gain in whom KDM4A expression was indeed lower than those who did not have this high risk cytogenetic abnormality. Moreover, KDM4A expression correlated adversely with expression of 1q21 genes (e.g., CKS1B, MCL1, PSMD4, ARNT). Whereas basal KDM4A protein level was moderately but clearly higher in MM cell lines carrying 1q21 gain or acquired drug resistance than their counterparts, exposure to hypoxia or lactic acid (but not cobalt chloride) resulted in marked KDM4A up-regulation, accompanied by NF-κB and HIF pathway activation. However, while NF-κB inhibition and to a lesser extent ARNT/HIF-1β knockdown led to a robust increase in hypoxia-induced KDM4A expression, shRNA knockdown or pharmacological inhibition of KDM4A triggered NF-κB activation and HIF expression, as well as up-regulated anti-apoptotic proteins (e.g., Mcl-1, TNFAIP3/A20, CKS1B), in association with increased H3K36me3 rather than H3K9me3. Furthermore, KDM4A knockdown or inhibition sharply diminished Btz lethality and overrode hypoxia-mediated cytoprotection. Interestingly, KDM4A knockdown also increased MM cell proliferation, promoted S phase entry, and attenuated Btz-induced DSB. Last, IHC of sequential bone marrow biopsies revealed that while KDM4A protein was relatively low at diagnosis, its level was markedly increased when patients achieved CR and then fell to the baseline low level at relapse. Conclusion: KDM4A/JMJD2A, a lysine demethylase that has been recognized as an pro-oncogenic protein via its epigenetic and/or non-epigenetic properties, is identified for the first time as a potential tumor suppressor in MM, particularly in a high risk subtype carrying 1q21 gain. Whereas KDM4A is expressed in MM and can be further induced by hypoxia that naturally exists in bone marrow niche, it seems to play multiple inhibitory roles in cell growth, cell cycle, DNA repair, and drug resistance by suppressing expression of oncogenic and anti-apoptotic genes (especially 1q21 genes), likely via H3K36me3 demethylation, and antagonizing NF-κB and HIF activation. These findings suggest that in contrast to its pro-oncogenic role in certain solid tumors, KDM4A might instead act as a tumor suppressor in MM. This work was supported by NNSFC (81471165, 81670189, and 81670190). Disclosures Kumar: AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Role of Hypoxia in the Progression and Dissemination of Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 421-421
Author(s):  
Abdel Kareem Azab ◽  
Phong Quang ◽  
Feda Azab ◽  
Aldo M. Roccaro ◽  
Antonio Sacco ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Stromal Cells ◽  
Research Funding ◽  
Phase Cell ◽  
Advisory Committees ◽  
Fluorescent Intensity ◽  
Hypoxic Conditions

Abstract Abstract 421 INTRODUCTION: Multiple myeloma (MM) is characterized by the disseminated involvement of the bone-marrow (BM), and its progression involves a continuous circulation of the MM cells in the peripheral blood and homing back to the BM. Several reports have described the mechanism involved in homing of MM cells to the BM. However, the driving force to metastasize from one site of the BM to another is not yet understood. Hypoxia (oxygen deprivation) was associated with poor patient prognosis in solid tumors, and several studies have shown that BM has a hypoxic nature. We hypothesize that the hypoxic nature of the BM and the rapid development of MM may cause induction of hypoxia in the tumor microenvironment, induce stress in the MM cells, and drive them to disseminate to new BM niches with normal oxygen levels (normoxia). METHODS AND RESULTS: First we have tested the level of oxygenation of MM cells and cells from its BM microenvironment by injection of pimonidazole (PIM) to mice with MM and determined the levels of PIM binding in MM cells and stromal cells in the microenvironment by flow cytometry. The whole cells population in the BM was hypoxic, while the MM cells were more hypoxic compared to cells in the microenvironment. We have mimicked these results in vitro by incubation of MM cells (cell lines and patients samples) in hypoxic conditions (0.5% oxygen, for 24 hrs). Induction of hypoxia was also verified by detection of increased binding of PIM by flow cytometry, and increased levels of HIF1 and HIF2 in MM cells by immunoblotting. MTT assay showed that hypoxia significantly decreased (40%) the survival of MM cells. Immunoblotting showed a downregulation of proliferative signaling pathway, including PI3K, AKT, mTOR and ERK; and induction of stress pathways including MKK-3 and 6, and p38. Apoptosis was not detected by flow cytometry after 24 hrs of hypoxic conditions. These results were confirmed by immunoblotting, which showed no change in apoptosis related proteins including caspases 3 and 7, Bcl-2, Bcl-xL and Mcl-1. However, hypoxia induced G1-phase cell cycle arrest (increase of 20% in G1)., These results were confirmed by immunoblotting showing downregulation of proteins associated with G1 to S phase transition including cyclins D1, D2, D3 and E1, and pRb; and upregulation of cell cycle inhibitor p27. Testing the effect of hypoxia on the adhesion properties of MM cells to BM stromal cells (BMSCs) revealed that hypoxic MM cells were 50% less adherent to BMSCs compared to normoxic MM cells. Also, hypoxic BMSCs induced 30% less adhesion of MM cells compared to normoxic BMSCs. Mechanistically, immunoblotting showed a significant decrease in the expression of cadherins in both hypoxic MM cells and hypoxic BMSCs. The chemokine stromal-cell derived factor-1-alpha (SDF1) is known to increase adhesive properties of MM cells, and to induce MM retention in the BM. Testing the levels of SDF1 revealed that hypoxia decreased SDF1 secretion (40%) from BMSCs. As a result, media from hypoxic BMSCs induce significantly decreased MM cell chemotaxis (20%) compared to media from normoxic BMSCs. These findings suggest that hypoxic BM will retain less MM cells in the BM and allow more egression. Comparison of the ability of hypoxic and normoxic MM cells to migrate towards normoxic BMSCs or SDF1 revealed that hypoxic MM cells had increased (250% and 350%, respectively) chemotactic properties compared to normoxic MM cells. These findings correlated with the increased expression of CXCR4 on hypoxic MM cells (mean fluorescent intensity shifts: Isotype=90, normoxic = 520, hypoxic = 1100). These results suggest that hypoxic MM cells will home to normoxic BM niches more efficiently than normoxic MM cells. Finally, testing the recovery of the hypoxic MM cells after incubation in normoxic conditions showed that the expression of CXCR4 was completely downregulated to levels found in normoxic cells after 6 hrs of exposure to normoxia. Moreover, incubation of hypoxic MM cells in normoxic BMSCs- media or SDF1 restored their expression of cadherins and their ability to adhere to BMSCs. CONCLUSION: In conclusion, we have showed that hypoxia inhibited the proliferation of MM cells and decreased their adhesion to BMSCs, as a first step of egress and metastasize to new normoxic BM niches. Disclosures: Anderson: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

scholarly journals Phase II Trial of the Combination of Ixazomib, Lenalidomide, and Dexamethasone in High-Risk Smoldering Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 804-804 ◽  
Author(s):  
Mark Bustoros ◽  
Chia-jen Liu ◽  
Kaitlen Reyes ◽  
Kalvis Hornburg ◽  
Kathleen Guimond ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
High Risk ◽  
Disease Progression ◽  
Board Of Directors ◽  
Rna Sequencing ◽  
Research Funding ◽  
Response Rate ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Background. This study aimed to determine the progression-free survival and response rate using early therapeutic intervention in patients with high-risk smoldering multiple myeloma (SMM) using the combination of ixazomib, lenalidomide, and dexamethasone. Methods. Patients enrolled on study met eligibility for high-risk SMM based on the newly defined criteria proposed by Rajkumar et al., Blood 2014. The treatment plan was designed to be administered on an outpatient basis where patients receive 9 cycles of induction therapy of ixazomib (4mg) at days 1, 8, and 15, in combination with lenalidomide (25mg) at days 1-21 and Dexamethasone at days 1, 8, 15, and 22. This induction phase is followed by ixazomib (4mg) and lenalidomide (15mg) maintenance for another 15 cycles. A treatment cycle is defined as 28 consecutive days, and therapy is administered for a total of 24 cycles total. Bone marrow samples from all patients were obtained before starting therapy for baseline assessment, whole exome sequencing (WES), and RNA sequencing of plasma and bone marrow microenvironment cells. Moreover, blood samples were obtained at screening and before each cycle to isolate cell-free DNA (cfDNA) and circulating tumor cells (CTCs). Stem cell collection is planned for all eligible patients. Results. In total, 26 of the planned 56 patients were enrolled in this study from February 2017 to April 2018. The median age of the patients enrolled was 63 years (range, 41 to 73) with 12 males (46.2%). Interphase fluorescence in situ hybridization (iFISH) was successful in 18 patients. High-risk cytogenetics (defined as the presence of t(4;14), 17p deletion, and 1q gain) were found in 11 patients (61.1%). The median number of cycles completed was 8 cycles (3-15). The most common toxicities were fatigue (69.6%), followed by rash (56.5%), and neutropenia (56.5%). The most common grade 3 adverse events were hypophosphatemia (13%), leukopenia (13%), and neutropenia (8.7%). One patient had grade 4 neutropenia during treatment. Additionally, grade 4 hyperglycemia occurred in another patient. As of this abstract date, the overall response rate (partial response or better) in participants who had at least 3 cycles of treatment was 89% (23/26), with 5 Complete Responses (CR, 19.2%), 9 very good partial responses (VGPR, 34.6%), 9 partial responses (34.6%), and 3 Minimal Responses (MR, 11.5%). None of the patients have shown progression to overt MM to date. Correlative studies including WES of plasma cells and single-cell RNA sequencing of the bone microenvironment cells are ongoing to identify the genomic and transcriptomic predictors for the differential response to therapy as well as for disease evolution. Furthermore, we are analyzing the cfDNA and CTCs of the patients at different time points to investigate their use in monitoring minimal residual disease and disease progression. Conclusion. The combination of ixazomib, lenalidomide, and dexamethasone is an effective and well-tolerated intervention in high-risk smoldering myeloma. The high response rate, convenient schedule with minimal toxicity observed to date are promising in this patient population at high risk of progression to symptomatic disease. Further studies and longer follow up for disease progression are warranted. Disclosures Bustoros: Dava Oncology: Honoraria. Munshi:OncoPep: Other: Board of director. Anderson:C4 Therapeutics: Equity Ownership; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Takeda Millennium: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghobrial:Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; BMS: Consultancy.

CYC065, a Potent Derivative of Seliciclib Is Active In Multiple Myeloma In Preclinical Studies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2999-2999 ◽  
Author(s):  
Samantha Pozzi ◽  
Diana Cirstea ◽  
Loredana Santo ◽  
Doris M Nabikejje ◽  
Kishan Patel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Preliminary Data ◽  
Research Funding ◽  
Preclinical Studies ◽  
Dependent Manner ◽  
Advisory Committees

Abstract Abstract 2999 Multiple myeloma (MM) is a treatable but incurable hematological malignancy and novel targeted therapies are under investigation. MM is characterized by dysregulation of the cell cycle, consequent to the overexpression of cyclins and their related kinases, the cyclins dependent kinases (CDK), a group of Ser/Thr proteine kinases. CDKs represent a promising therapeutic target, and inhibitors have been developed for anticancer treatment. We have previously studied seliciclib in the context of MM. CYC065, a second generation CDK inhibitor is the more potent derivative of seliciclib. It is mainly active on CDK 2, 5 and 9, involved in progression of the cell cycle and protein transcription. It has already shown promising results in preclinical studies in breast cancer and acute leukemia. We tested CYC065 in in vitro experiments in MM. Our preliminary data in 7 MM cell lines showed cytotoxicity of CYC065, both in MM cell lines sensitive as well as resistant to conventional chemotherapy, with an IC50 ranging between 0.06 and 2μ M, at 24 and 48h. Tritiated thymidine uptake assay confirmed the antiproliferative effects of CYC065 in MM, and its ability to overcome the growth advantage conferred by co-culture with bone marrow stromal cells derived from MM patients, and cytokines like interleukin 6 (10ng/ml) and insulin like growth factor-1 (50ng/ml). The anti-proliferative effect was evident both at 24 and 48h, starting at concentrations as low as 0.015μ M. The AnnexinV/PI assay in the MM1.s cell line confirmed CYC065's ability to induce apoptosis in a time dependent manner starting at 9 hours of treatment, at a concentration of 0.125 μ M, inducing 82% of apoptosis after 48h of exposure. Cell cycle analysis in the same MM1.s cell line showed an increase of subG1 phase, starting at 9 hours of treatment, at 0.125 μ M of CYC065. Preliminary results of western blot analysis confirmed the apoptotic effect of CYC065 in the MM1s cell line, highlighted by the cleavage of caspase 3, 8, 9 and PARP. The compound was tested in primary CD138+ cells isolated from three refractory MM patients, confirming its efficacy at 0.125 μ M, both at 24 and 48h. Comparative analysis in PBMCs from normal donors, for the evaluation of the drug toxicity is ongoing and will be presented. In conclusion our preliminary data confirm the efficacy of CYC065 in MM cell lines and primary MM cells, at nanomolar concentrations. Ongoing mechanistic and in vivo studies will delineate its role in the now increasing spectrum of CDK inhibitors in MM and better define its potential for clinical development in MM. Disclosures: Green: Cyclacel: Employment. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Acetylon: Research Funding.

Investigational Agent MLN9708 Target Tumor Suppressor MicroRNA-33b in Multiple Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 136-136
Author(s):  
Ze Tian ◽  
Jian-Jun Zhao ◽  
Jianhong Lin ◽  
Dharminder Chauhan ◽  
Kenneth C. Anderson
Keyword(s):  
Multiple Myeloma ◽  
Tumor Suppressor ◽  
Board Of Directors ◽  
Expression Profiling ◽  
Research Funding ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Investigational Agent ◽  
Reporter Activity

Abstract Abstract 136 Investigational Agent MLN9708 Target Tumor Suppressor MicroRNA-33b in Multiple Myeloma Cells Ze Tian, Jianjun Zhao, Jianhong Lin, Dharminder Chauhan, Kenneth C. Anderson Medical Oncology, Dana Farber Cancer Institute and Harvard Medical School, Boston, MA, 02115 MicroRNAs (miRNAs) are 19–25 nucleotide-long noncoding RNA molecules that regulate gene expression both at the level of messenger RNA degradation and translation. Emerging evidence shows that miRNAs play a critical role in tumor pathogenesis by functioning as either oncogene or tumor suppressor genes. The role of miRNA and their regulation in response to proteasome inhibitors treatment in Multiple Myeloma (MM) is unclear. Here, we utilized MLN9708, a selective orally bio-available proteasome inhibitor to examine its effects on miRNA alterations in MM.1S MM cells. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to its biologically active form MLN2238. Our previous study using both in vitro and in vivo models showed that MLN2238 inhibits tumor growth and triggers apoptosis via activation of caspases. Moreover, MLN2238 triggered apoptosis in bortezomib-resistant MM cells, and induced synergistic anti-MM activity when combined with HDAC inhibitor SAHA, dexamethasone, and lenalidomide. In the current study, we treated MM.1S cells with MLN2238 (12 nM) for 3 hours and harvested; total RNA was subjected to miRNA profiling using TaqMan® Array Human miRNA A-Card Set v3.0 and the data was analyzed using dChip analysis. Results showed that MLN2238 modulates miRNA expression with a total of 36 miRNA changing their expression profiling (δδCT>1.5 or δδCT <-1.5; 19 were upregulated and 17 showed a downregulation). Among all miRNA, miR-33b was highly (δδCT>7) upregulated in response to MLN2238 treatment. We therefore hypothesized that miR-33b may play a role in MM pathogenesis as well as during MLN2238-induced proteasome inhibition in MM cells. We first utilized quantitative polymerase chain reaction (q-PCR) to validate the changes in miRNA expression profiling. Results confirmed that MLN2238 treatment triggers significant increase in the miR-33b expression in MM.1S cells (2.1 and 2.2 folds at 3h and 6h, respectively; P<0.001). Examination of normal PBMCs and plasma cells showed higher expression of miR-33b than patient MM cells (P<0.001). We further investigated the functional role of miR-33b in MM cells at baseline and during MLN2238 treatment. Drug sensitivity, cell viability, apoptosis, colony formation, and migration assays were performed using cell TilTer-Glo, Annexin V-FITC/PI staining, MTT staining, and Transwell assays, respectively. Signaling pathways modulated post miR-33b overexpression were evaluated by q-PCR, immunoblot, and reporter assays. Our findings show that overexpression of miR-33b significantly decreased cell viability, cell migration, colony formation, as well as increased apoptosis and sensitivity of MM cells to MLN2238 treatment. Targetscan analysis predicted pim-1 as a putative downstream target of miR-33b. Overexpression of miR-33b downregulated pim-1 mRNA and protein expression. To further corroborate these data, we co-tranfected miR-33b and Pim-1-wt or Pim-1-mt in 293T and MM.1S cell lines. In concert with our earlier findings, miR-33b decreases pim-1-wt, but not pim-1-mt reporter activity in both cell lines. Reflecting the overexpression study results, MLN2238 treatment also decreases pim-1-wt, but not pim1-mt reporter activity. Moreover, a biochemical inhibitor of pim1/2 triggered apoptosis in MM cells. Finally, overexpression of miR-33b inhibits tumor growth (P<0.001) and prolongs survival (P<0.001) in both subcutaneous and disseminated human MM xenograft models. In summary, our study suggests that miR-33b is a tumor suppressor, which plays a role during MLN2238-induced apoptotic signaling in MM cells, and provide the basis for novel therapeutic strategies targeting miR-33b in MM. Disclosures: Anderson: Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership.

Metabolomic Profiling Identifies Mechanisms Regulating Hypoxia-Induced Drug Resistance in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3944-3944
Author(s):  
Patricia Maiso ◽  
Yosra Aljawai ◽  
Antonio Sacco ◽  
Susanne B Breitkopf ◽  
Ilyas Sahin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Drug Resistance ◽  
High Risk ◽  
Tumor Cells ◽  
Advisory Board ◽  
Water Soluble ◽  
Parp Cleavage ◽  
Hypoxic Conditions ◽  
Bortezomib Treatment

Abstract Abstract 3944 Introduction: Multiple Myeloma (MM) is the second most prevalent hematological malignancy and remains incurable, with a median survival of 3–7 years. However, despite the success of the new treatments, most patients still succumb to their disease. In about 20–25% of high-risk patients, MM progresses rapidly and does not respond to conventional therapies leading to rapid extramedullary disease and demise of these patients. One such regulator of dissemination and drug resistance is the dynamic process of oxygen deprivation or hypoxia. A number of studies show that hypoxia promotes neo-angiogenesis, cancer progression, epithelial-mesenchymal transition (EMT), acquisition of metastasis potential and stem-cell features, as well as resistance to therapy by activating adaptive transcriptional programs. Targeting hypoxia, and the metabolic pathways regulated by hypoxia in the tumor cells, could lead to novel opportunities for cancer therapy. Rapidly proliferating hypoxic cancer cells undergo a “metabolic switch” to anaerobic glycolysis. This altered energy metabolism has been shown to be associated with activated oncogenes and mutant tumor suppressors, which are more prevalent in patients with high-risk MM. Methods: The effect of hypoxia was analyzed in different MM cell lines (MM1S, RPMI8226, U266 and H929) in basal conditions and after the treatment with bortezomib, dexamethasone or melphalan. The cytotoxicity was analyzed by means of MTT assay. Cell cycle and apoptosis studies were performed by flow cytometry. Proteomic changes induced after treatment were analyzed under normoxic and hypoxic conditions by western-blotting. Gene expression profile of MM1S cells treated with bortezomib was compared in normoxia vs hypoxia using D-chip software. Genes with expression changes greater or lower than 2 fold in either direction were selected. HIF1A and HIF2A knockdowns were performed in MM1S using lentiviral vectors. For metabolite collection, samples were re-suspended using HPLC grade water for mass spectrometry and analyzed using a 5500 QTRAP hybrid triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC system (Shimadzu). A total of 254 endogenous water soluble metabolites were analyzed. Results: We observed that hypoxic conditions (12 hours at 0.7% of oxygen levels) suppressed the effect of melphalan and more significantly the effect of bortezomib. At the transcriptional level and protein level, we observed that cells treated with bortezomib in hypoxic conditions affected a large number of genes/proteins involved in cell cycle, cell death, glucose metabolism and the Wnt signaling pathway. Hypoxia blocked cell cycle progression, which was accompanied by p21, p53 and p57 up-regulation. In addition, apoptosis pathways were inhibited after exposure to hypoxia including inactivation of caspases 3, 8 and 9 and PARP cleavage. HIF1A and HIF2A knockdowns restore the effect of bortezomib in MM1S and increased the percentage of apoptosis in cells treated with bortezomib under hypoxic conditions. To further explore the role of hypoxia in the regulation of tumor metabolism, metabolomic studies were performed to characterize metabolic alterations following bortezomib treatment. This analysis revealed that hypoxic tumor cells treated with bortezomib show significant metabolic changes involving multiple pathways, the most significant of which are intermediates in glucose and, sucrose metabolism. Bortezomib treatment under hypoxic conditions was accompanied by a significant decrease in UDP-D-glucose, UDP-D-glucuronate, and glutathione disulfide. Conclusion: Hypoxic conditions are essential for drug resistance and glucose utilization. These data provide new therapeutic targets and associated biomarkers for the treatment of Multiple Myeloma. Disclosures: Ghobrial: Millennium: Advisory Board Other; Novartis: Advisory Board, Advisory Board Other.

scholarly journals Effect of the Wnt-Signaling Inhibitor Sclerostin and Bone Marrow Adipocytes on Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2077-2077
Author(s):  
Cristina Panaroni ◽  
Ka Tat Siu ◽  
Keertik S Fulzele ◽  
Janani Ramachandran ◽  
Noopur Raje
Keyword(s):  
Multiple Myeloma ◽  
Wnt Signaling ◽  
Board Of Directors ◽  
Research Funding ◽  
Neutralizing Antibody ◽  
Fold Increase ◽  
Advisory Committees ◽  
Cell Engraftment ◽  
Msc Differentiation

Abstract Multiple Myeloma (MM) is a plasma cell tumor that originates and expands within bone marrow (BM). MM occurs primarily later in life with a median age at diagnosis in the seventh decade characterized by loss of bone tissue due to osteolytic lesions. Recent studies have suggested a positive correlation of MM with obesity. BM fat cells originate, as bone cells, from a common progenitor called Mesechymal Stem Cell (MSC). MSC commitment into osteoblastic or adipose lineage is regulated by two major signaling pathways: Wnt signaling, which promotes bone formation, and PPARγ signaling, responsible for adipocyte differentiation. Adipogenesis and osteogenesis are inversely correlated processes, thus a signaling imbalance favoring differentiation towards one lineage will tilt this critical balance. In fact, the amount of marrow adiposity increases with age such that it is 30% of the BM volume in young adults but rises up to 70% in elderly people while bone formation reduces with age. We have recently demonstrated that MM patients have increased levels of a Wnt-signaling inhibitor sclerostin (SOST) and patient MSC differentiation into osteoblastic cells is improved in the presence of a SOST neutralizing antibody (Eda et al. JBMR 2015). Sclerostin is secreted by osteolineage cells and has also been shown to increase adipogenesis of an adipogenic cell line 3T3-L1 (Ukita et al. JCB 2016). However, the role of SOST on BM adipose tissue in MM patients has not been investigated. Here we show that elevated SOST levels, induced by MM cells, increase BM adipogenesis which, in turn, supports MM progression. To assess the role of MM cells on MSC differentiation into adipocytes, MSCs from patients and normal donors (ND) were differentiated in vitro in the presence or absence of MM.1S, a human MM cell line. Presence of MM.1S cells significantly reduced osteogenic differentiation of MSCs as assessed by quantitative and qualitative Alizarin Red S staining; by contrast, the presence of MM.1S cells significantly enhanced adipogenesis in the MSCs as assessed by Oil Red O staining quantification. At the molecular level, we observed a 2-fold increase in PPARγ gene expression in MSCs from MM patients when compared to ND samples at baseline and a 5-fold increase when MSCs from patients and NDs were cultured for 72 hours in the presence of MM.1S in trans-well. To evaluate the role of SOST on BM adiposity, we intraperitoneally injected recombinant SOST, or PBS as control, into wild-type (WT) mice daily for 3 weeks. At the end of the treatment, mice treated with SOST showed a significant increase in BM adiposity. To validate MM cell induced adipogenesis in in-vivo settings, we injected MM.1S cells into the SCID-hu immunodeficient mouse model. Four weeks after cell injection, increased BM adipocytes were observed in MM.1S treated immunodeficient mice. Interestingly, when mice injected with MM.1S cells were treated for 3 weeks with SOST neutralizing antibody the level of BM adiposity returned to the healthy control levels. Finally, MM cell engraftment and tumor development was analyzed in SOST knock-out (KO) mice. While both SOST KO and WT animals showed MM cell engraftment and extramedullary plasmacytoma formation, preliminary results suggest a lower level of MM cell engraftment in BM of SOST KO mice. Our data suggest that sclerostin secretion increases BM adipogenesis supporting MM cell growth and survival and therefore may play a critical role in the development and progression of MM. Disclosures Raje: Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Eli Lilly: Research Funding.

scholarly journals Multiple Myeloma Pathogenesis: The Role of Junb in Bone Marrow Angiogenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4341-4341
Author(s):  
Fengjuan Fan ◽  
Stefano Malvestiti ◽  
Yujia Shen ◽  
Eugenio Morelli ◽  
Yuji Mishima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Research Funding ◽  
Angiogenic Factors ◽  
University Research ◽  
Advisory Committees ◽  
Cell Clones ◽  
In Vitro Angiogenesis

A significant increase in bone marrow (BM) angiogenesis represents a key event in early, microenvironment-dependent, multiple myeloma (MM). Angiogenic growth factor- and cytokine- production and secretion is a complex process regulated by a plethora of transcription factors (TFs). Over the past years, members of the AP-1 family of TFs have emerged as potential new therapeutic targets. Our recent work demonstrated for the first time a pivotal role for the AP-1 family member JunB in MM pathogenesis (Fan et al., 2017). Whether JunB also contributes to MM BM angiogenesis is currently unknown. In silico and immunohistochemical analyses revealed a correlative increase of JunB and angiogenic growth factors in samples isolated from healthy donors to MGUS and MM patients; and a decrease in samples isolated from patients with plasma cell leukemia. These data were supported by the utilization of an innovative in vivo MM model of clonal evolution. Specifically, JunB as well as selected angiogenic factors were significantly increased in tumor cell clones at primary sites (bone chips) versus tumor cell clones at metastatic (distant BM) sites, as evidenced by whole exome and RNA sequencing. Functionally, doxycyclin- induced inhibition of stroma cell: MM cell co-culture- as well as of IL-6- mediated JunB upregulation in TetR-shJunB/ MM.1S cells significantly reduced production and secretion of angiogenic factors; and consequently inhibited in vitro angiogenesis. Conversely, 4-hydroxytamoxifen (4-OHT)-mediated upregulation of JUNB activity in JUNB-ER/MM cells strongly increased the expression and secretion of angiogenic factors and in vitro angiogenesis. The interaction of JunB with angiogenic factor- encoding DNA in MM cells was further confirmed utilizing chromatin immunoprecipitation (ChIP)- sequencing. Finally, treatment with doxycycline effectively inhibited JunB levels and consistently reduced microvessel density in immunodeficient NSG mice inoculated with TetR-shJUNB/ MM.1S, but not TetR-SCR/ MM.1S. In conclusion, our findings demonstrate a pivotal role of JUNB in MM BM angiogenesis; they thereby provide further evidence that JUNB is a promising therapeutic target particularly in early MM. Disclosures Vallet: Pfizer: Honoraria; Roche Pharmaceuticals: Consultancy; MSD: Honoraria. Roccaro:Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; AstraZeneca: Research Funding; Transcan2-ERANET: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Transcan2-ERANET: Research Funding; AstraZeneca: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; European Hematology Association: Research Funding; European Hematology Association: Research Funding. Goldschmidt:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; John-Hopkins University: Research Funding; Chugai: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Molecular Partners: Research Funding. Podar:Takeda: Consultancy; Celgene: Consultancy, Honoraria; Amgen Inc.: Honoraria; Janssen Pharmaceuticals: Consultancy, Honoraria; Roche Pharmaceuticals: Research Funding.

scholarly journals Genomic Profiling of Smoldering Multiple Myeloma Classifies Molecular Groups with Distinct Pathogenic Phenotypes and Clinical Outcomes

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 723-723
Author(s):  
Shankara Anand ◽  
Mark Bustoros ◽  
Romanos Sklavenitis-Pistofidis ◽  
Robert A. Redd ◽  
Eileen M Boyle ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
High Risk ◽  
Board Of Directors ◽  
Research Funding ◽  
Molecular Classification ◽  
Intermediate Risk ◽  
Advisory Committees ◽  
Increased Risk ◽  
Risk Of Progression ◽  
Over Time

Abstract Introduction: Multiple Myeloma (MM) is an incurable plasma cell malignancy commonly preceded by the asymptomatic stage smoldering multiple myeloma (SMM). MM is characterized with significant genomic heterogeneity of chromosomal gains and losses (CNVs), translocations, and point mutations (SNVs); alterations that are also observed in SMM patients. However, current SMM risk models rely solely on clinical markers and do not accurately capture progression risk. While incorporating some genomic biomarkers improves prediction, using all MM genomic features to comprehensively stratify patients may increase risk stratification precision in SMM. Methods: We obtained a total of 214 patient samples at SMM diagnosis. We performed whole-exome sequencing on 166 tumors; of these, RNA sequencing was performed on 100. Targeted capture was done on 48 additional tumors. Upon binarization of DNA features, we performed consensus non-negative matrix factorization to identify distinct molecular clusters. We then trained a random forest classifier on translocations, SNVs, and CNVs. The predicted clinical outcomes for the molecular subtypes were further validated in an independent SMM cohort of 74 patients. Results: We identified six genomic subtypes, four with hyperdiploidy (&gt;48 chromosomes, HMC, HKR, HNT, HNF) and two with IgH translocations (FMD, CND) (Table 1). In multivariate analysis accounting for IMWG (20-2-20) clinical risk stages, high-risk (HMC, FMD, HKR) and intermediate-risk (HNT, HNF) genetic subtypes were independent predictors of progression (Hazards ratio [HR]: 3.8 and 5.5, P = 0.016 and 0.001, respectively). The low-risk, CND subtype harboring translocation (11;14) was enriched for the previously defined CD-2 MM signature defined by the B cell markers CD20 and CD79A (FDR = 0.003 ), showed upregulation of CCND1, E2F1, and E2F7 (FDR = 0.01, 0.0004, 0.08), and was enriched for G2M checkpoint, heme metabolism, and monocyte cell signature (FDR = 0.003, 0.003, 0.003, respectively). The FMD subtype with IgH translocations (4;14) and (14;16) was enriched for P53, mTORC1, unfolded protein signaling pathways and plasmacytoid dendritic cell signatures (FDR = 0.01, 0.005, 0.008, respectively). The HKR tumors were enriched for inflammatory cytokine signaling, MYC target genes, T regulatory cell signature, and the MM proliferative (PR) signatures (FDR = 0.02, 0.03, 0.007, 0.02, respectively). The APOBEC mutational signature was enriched in HMC and FMD tumors (P = 0.005), while there was no statistical difference across subtypes in the AID signature. The median follow-up for the primary cohort is 7.1 years. Median TTP for patients in HMC, FMD, and HKR was 3.8, 2.6, and 2.2 years, respectively; TTP for HNT and HNF was 4.3 and 5.2, respectively, while it was 11 years in CND patients (P = 0.007). Moreover, by analyzing the changes in MM clinical biomarkers over time, we found that patients from high-risk subgroups had higher odds of developing evolving hemoglobin and monoclonal protein levels over time (P = 0.01 and 0.002, respectively); Moreover, the absolute increase in M-protein was significantly higher in patients from the high-risk genetic subtypes at one, two, and five years from diagnosis (P = 0.001, 0.03, and 0,01, respectively). Applying the classifier to the external cohort replicated our findings where intermediate and high-risk genetic subgroups conferred increased risk of progression to MM in multivariate analysis after accounting for IMWG staging (HR: 5.5 and 9.8, P = 0.04 and 0.005, respectively). Interestingly, within the intermediate-risk clinical group in the primary cohort, patients in the high-risk genetic subgroups had increased risk of progression (HR: 5.2, 95% CI 1.5 - 17.3, P = 0.007). In the validation cohort, these patients also had an increased risk of progression to MM (HR: 6.7, 95% CI 1.2 - 38.3, P = 0.03), indicating that molecular classification improves the clinical risk-stratification models. Conclusion: We identified and validated in an independent dataset six SMM molecular subgroups with distinct DNA alterations, transcriptional profiles, dysregulated pathways, and risks of progression to active MM. Our results underscore the importance of molecular classification in addition to clinical evaluation in better identifying high-risk SMM patients. Moreover, these subgroups may be used to identify tumor vulnerabilities and target them with precision medicine efforts. Figure 1 Figure 1. Disclosures Bustoros: Janssen, Bristol Myers Squibb: Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria. Casneuf: Janssen: Current Employment. Kastritis: Amgen: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Genesis Pharma: Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Walker: Bristol Myers Squibb: Research Funding; Sanofi: Speakers Bureau. Davies: Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Dimopoulos: Amgen: Honoraria; BMS: Honoraria; Takeda: Honoraria; Beigene: Honoraria; Janssen: Honoraria. Bergsagel: Genetech: Consultancy, Honoraria; Oncopeptides: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Patents & Royalties: human CRBN mouse; GSK: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Yong: BMS: Research Funding; Autolus: Research Funding; Takeda: Honoraria; Janssen: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; GSK: Honoraria; Amgen: Honoraria. Morgan: BMS: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees. Getz: IBM, Pharmacyclics: Research Funding; Scorpion Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy.

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